Inhibition of il-17 production

ABSTRACT

The invention concerns inhibition of the production of proinflammatory cytokine interleukin-17 (IL-17) by T cells, using an antagonist of interleukin-23 (IL-23). The invention further concerns the use of IL-23 antagonists in the treatment of inflammatory diseases characterized by the presence of elevated levels of IL-17. IL-23 antagonists include, without limitation, antibodies specifically binding to a subunit or IL-17 or an IL-17 receptor. The invention additionally concerns induction of IL-7 production by using an IL-23 agonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35U.S.C. §120 to U.S. patent application Ser. No. 12/350,125, filed Jan.7, 2009, which is a divisional of, and claims priority under 35 U.S.C.§120 to, U.S. patent application Ser. No. 10/697,599, filed Oct. 29,2003 (now U.S. Pat. No. 7,510,709) which claims priority under U.S.C.§119(e) to U.S. Provisional Patent Application Ser. No. 60/423,090 filedOct. 30, 2002, the entireties of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns inhibition of the production ofproinflammatory cytokine interleukin-17 (IL-17) by T cells, using anantagonist of interleukin-23 (IL-23). The invention further concerns theuse of IL-23 antagonists in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17.

2. Description of the Related Art

IL-17 is a T cell derived pro-inflammatory molecule that stimulatesepithelial, endothelial and fibroblastic cells to produce otherinflammatory cytokines and chemokines including IL-6, IL-8, G-CSF, andMCP-1 (S. Aggarwal, A. L. Gurney, J Leukoc Biol 71, 1 (2002); Z. Yao etal., Immunity 3, 811 (1995); J. Kennedy et al., J Interferon CytokineRes 16, 611 (1996); F. Fossiez et al., J Exp Med 183, 2593 (1996); A.Linden, H. Hoshino, M. Laan, Eur Respir J 15, 973 (2000); X. Y. Cai, C.P. Gommoll, Jr., L. Justice, S. K. Narula, J. S. Fine, Immunol Lett 62,51 (1998); D. V. Jovanovic et al., J Immunol 160, 3513 (1998); and M.Laan et al., J Immunol 162, 2347 (1999)).

IL-17 also synergizes with other cytokines including TNF-α and IL-1β tofurther induce chemokine expression (Jovanovic et al., supra, and M.Chabaud, F. Fossiez, J. L. Taupin, P. Miossec, J Immunol 161, 409(1998)). Levels of IL-17 are found to be significantly increased inrheumatoid arthritis (RA) synovium (S. Kotake et al., J Clin Invest 103,1345 (1999); and M. Chabaud et al., Arthritis Rheum 42, 963 (1999)),during allograft rejection (M. A. Antonysamy et al., Transplant Proc 31(1999); M. A. Antonysamy et al., J Immunol 162, 577 (1999); C. C. Loong,C. Y. Lin, W. Y. Lui, Transplant Proc 32 (2000); and H. G. Hsieh, C. C.Loong, W. Y. Lui, A. Chen, C. Y. Lin, Transpl Int 14, 287 (2001)), andin other chronic inflammatory diseases including multiple sclerosis (K.Kurasawa et al., Arthritis Rheum 43, 2455 (2000)) and psoriasis (C.Albanesi et al., J Invest Dermatol 115, 81 (2000), and B. Homey et al.,J Immunol 164, 6621 (2000)). Although clearly produced by activated Tcells, previous reports have not provided clear classification of IL-17within the paradigm of Th1 and Th2 polarized cytokine profiles.

IL-23 is a heterodimeric cytokine, sharing a subunit, termed p40, withinterleukin-12 (IL-12), that combines with a unique subunit, p19 (B.Oppmann et al., Immunity 13, 715 (2000)). IL-23 has been reported topromote the proliferation of T cells, in particular memory T cells (D.M. Frucht, Sci STKE 2002 Jan. 8; 2002(114):PE1). Transgenic p19 micehave been recently described to display profound systemic inflammationand neutrophilia (M. T. Wiekowski et al., J Immunol 166, 7563 (2001)).

No correlation has so far been established between the expression andbiological roles of the IL-17 and IL-23 cytokines.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method for inhibitinginterleukin-17 (IL-17) production by T cells comprising treating the Tcells with an antagonist of interleukin-23 (IL-23).

In another aspect, the invention concerns a method for the treatment ofan inflammatory disease characterized by elevated expression ofinterleukin 17 (IL-17) in a mammalian subject, comprising administeringto the subject an effective amount of an antagonist of interleukin-23(IL-23).

In yet another aspect, the invention concerns a method for identifyingan anti-inflammatory agent comprising the steps of:

(a) incubating a culture of T cells with IL-23, in the presence andabsence of a candidate molecule;

(b) monitoring the level of IL-17 in the culture; and

(c) identifying the candidate molecule as an anti-inflammatory agent ifthe level of IL-17 is lower in the presence than in the absence of suchcandidate molecule.

In a further aspect, the invention concerns a method for inducing IL-17production in a mammalian subject comprising administering to saidsubject an IL-23 agonist.

In all aspects, the antagonist or agonist preferably is an anti-IL-23 oranti-IL-23 receptor antibody, including antibody fragments. Theinflammatory disease preferably is a chronic inflammatory condition,such as, for example, rheumatoid arthritis (RA), graft versus hostreaction that may lead to allograft rejection, multiple sclerosis (MS)or psoriasis. The induction of IL-17 production is typically useful inpatients subjected to bacterial infection, such as, for example,infection Mycobacterium tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: IL-17 production in different cell types:

FIG. 1 panel A shows single cell suspensions of spleen were preparedfrom C57/BL-6 mice and mononuclear cells were isolated from suspendedsplenocytes by density gradient centrifugation. 2×10⁶ cells/ml werecultured in the presence or absence of microbial lipopeptide LBP (100ng/ml), LPS (100 ng/ml) or LTA (100 ng/ml) for 3 days, following whichthe cells were collected and analyzed for IL-17 using ELISA.

FIG. 1 panel B shows purified T cells were obtained from murinesplenocytes following positive selection of FACS sorted CD90 labeledcells. These cells were cultured (1×106 cells/ml) in presence or absenceof plate-bound anti-CD3 (5 μg/ml), or supernatant from activateddendritic cells (LPS-treated) for 3 days and culture supernatantscollected and analyzed for IL-17 levels using ELISA kit. Dendritic cellswere derived from macrophages (obtained as adherent population fromsplenocyte suspension), by treating macrophages with rmGM-CSF (2 ng/ml)and rmIL-4 (1000 U/ml) for 4 days, washing and re-activating using LPS(0.5 μg/ml). Representative results from 3 independent experiments areshown.

FIGS. 2A and 2B: IL-23 stimulates production of IL-17:

FIG. 2A shows mononuclear cells isolated from splenocytes were cultured(2×10⁶ cells/ml) with 100 U/ml recombinant IL-2 and were incubated inpresence or absence of various concentrations of IL-23 (0.1-1000 ng/ml)for 6 days. Levels of IL-17 accumulated in culture supernatants weremeasured using ELISA

FIG. 2B shows changes in mRNA levels for IL-17 in response to IL-23treatment were measured by quantitative RT-PCR. Plotted is the relativechange in Ct (cycle threshold) of the PCR reaction. Data for each sampleis normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA levelpresent in each sample and then normalized again between samples to thelevel of IL-17 mRNA present in the time zero unstimulated conditions. Aseach Ct corresponds to a PCR cycle, one Ct is approximately equal to a2-fold change in mRNA abundance. The approximate mRNA fold differencefor 5 Ct and 10 Ct changes are indicated in parenthesis. The experimentwas performed with splenocytes from 4 mice, and the individual datapoints are represented with x and the average Ct change is indicated bybar columns

FIG. 2C shows changes in mRNA levels of the IL-17 family member IL-17Fin response to IL-23 treatment were measured by quantitative RT-PCR asin the legend to FIG. 2B.

FIG. 3: IL-23 acts on memory T cells to induce IL-17 production:

FIG. 3 shows mononuclear cells isolated from single cell suspension ofmurine splenocytes were stained with (a) CyC-CD4+PE-CD44 or (b)CyC-CD4+PE-CD62L and sorted for CD4⁺ cells that were eitherCD44^(high)/CD62L^(low) for memory phenotype or CD44^(low)/CD62^(high)for naïve phenotype. The sorted cells were cultured with 100 U/MLrecombinant IL-2 in the presence or absence of IL-23 (or its boiled prepas a control), plate bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml)for 5 days, washed, and re-stimulated with anti-CD3 antibody for another24 hours. Supernatants were collected and IL-17 levels were measuredusing ELISA.

FIGS. 4A and 4B: IL12p40 antibody blocks IL-23-dependent IL-17production:

FIG. 4A shows increasing concentrations of p40 antibody or an unrelatedisotype-matched control antibody were pre-incubated with IL-23 (100ng/ml) for 1 hr. at 37° C. and then incubated for another 5-6 days withmononuclear cells isolated from mouse spleen (2×10⁶ cells/ml) inpresence of recombinant IL-2. Supernatant were harvested and levels ofIL-17 measured using ELISA (left panel). Optimum concentrations ofIL-12p40 antibody or an unrelated isotype-matched control antibody werepre-incubated with conditioned media of LPS-stimulated dendritic cells(10% v/v) for 1 hr. at 37° C. and then incubated for another 5 days withmononuclear cells isolated from mouse spleen (2×10⁶ cells/ml) inpresence of recombinant IL-2. Supernatant were harvested and levels ofIL-17 measured using ELISA (right panel).

FIG. 4B shows mononuclear cells isolated from splenocytes of wild typemice (C57/BL6) or mice lacking one of the components of IL-12, i.e.IL12a^(−/−) (p35 knockout) or IL12b^(−/−) (p40 knockout) were culturedin the presence of ConA for 3 days and IL-17 levels measured insupernatants using ELISA.

FIGS. 5A and 5B: effect of IL-12 on IL-17 production:

FIG. 5A shows mononuclear cells isolated from spleen cell cultures wereincubated in the presence purified IL-23 (1 nM) and the indicatedconcentration of IL-12 for 5 days and then washed and re-stimulated withConA for another 24 hours. IL-17 levels were measured in cellsupernatant using ELISA kits.

FIG. 5B shows mononuclear cells isolated from spleen cell cultures fromwild type or mice lacking IL-12Rβ2 (IL-12Rβ2^(−/−) ko) were incubated inthe presence or absence of purified IL-23 (1 nM) for 5 days and thenwashed and re-stimulated with ConA for another 24 hours. IL-17 and IFN-γlevels were measured in cell supernatant using ELISA kits.

FIGS. 6A, 6B and 6C: targeting of the IL-23p19 locus:

FIG. 6A shows native IL-23p19 locus (top), the targeting construct(middle), and the correctly targeted locus (bottom) are depicted toscale unless otherwise indicated by double slashes. Open boxes indicatecoding exons, and hatched boxes represent exons encoding 5′ and 3′untranslated regions of the resulting messenger RNA (mRNA). The fourcoding exons of the p19 gene are numbered. Boxes with arrows indicatethe promoter regions for neomycin (neo) and thymidine kinase (tk)selection cassettes, and an open box labeled EGFP indicates the locationof an enhanced green fluorescent protein reporter gene. Restrictionsites used for cloning and analysis of the arms are labeled as follows:B, Bam HI; S, Sac II; E, Eco RI; Bg, Bgl II; X, Xho I. The location ofan antisense primer used to amplify the short arm is indicated by theletter P and an arrow. The size of restriction fragments resulting fromdigestion with Bam HI and Eco RI are indicated in the wild type (WT) andthe mutated (MUT) locus, and the locations of two probes used to detectthese fragments by southern blot are shown by thick lines.

FIGS. 6B and 6C: Southern blot analysis of Bam HI digests probed withprobe 1, and Eco RI digests probed with probe II, respectively. DNA wasextracted from wild-type (WT) embryonic stem (ES) cells, from ES clone1c5, and from a wild type, a heterozygous (HET), and a knockout (KO)mouse. The identity of the band is indicated at the left side of theblot, while its size is given on the right side.

FIG. 7: Total serum immunoglobulin levels IL-23p19^(−/−) mice. Serumlevels of immunoglobulin isotypes were determined by isotype specificELISA from groups of 16 wild type (filled circles) and IL-23p19^(−/−)(open circles) mice. Immunoglobulin isotypes are indicated at the bottomof the graph.

FIGS. 8A-8F: Humoral immune response in IL-23p19^(−/−) mice. A-F:Ovalbumin (OVA) specific levels of IgG1 (FIG. 8A), IgG2a (FIG. 8B),IgG2b (FIG. 8C), IgG3 (FIG. 8D), IgE (FIG. 8E), and IgA (FIG. 8F) afterone (1^(st)) and two (2^(nd)) immunizations with OVA. Filled circles,wild-type mice; open circles, and IL-23p19^(−/−) mice, gray circles, andIL-12p40^(−/−) mice. Arbitrary units were calculated as described inmethods and materials. The average of each group is indicated by both ablack horizontal bar and a numeric value at the bottom of the graph.Asterisks mark statistically significant P-values of less than 0.05.

FIG. 9: T-independent B-cell responses are normal in IL-23p19^(−/−)mice. Serum levels of TNP specific IgM was determined by ELISA from miceimmunized with TNP-LPS (type I, left) or TNP-Ficoll (type II, right).Filled circles, wild-type mice; open circles, and IL-23p19^(−/−) mice.

FIGS. 10A and 10B: Memory T-cell function. Wild type (filled circles)and IL-23p19^(−/−) mice (open circles) were immunized on day 0 withOvalbumin and challenged on day 21 with TNP-OVA. Serum was harvested ondays 0, 14, and 26 and tested by ELISA for the presence of TNP-specificIgG1 (FIG. 10A) and IgG2a (FIG. 10B). For IgG1, a commercially availablestandard was used. For IgG2a, arbitrary units were calculated asdescribed in methods and materials.

FIG. 11: Delayed type hypersensitivity (DTH) reactions. Antigen specificswelling is calculated as percent increase in footpad thickness over thevalue measured just before the challenge. The results were averaged overall six mice in each group, and error bars represent the standarddeviations. A second wild-type group that was not sensitized is used asa control for swelling induced by the antigen alone. An asterisk insidea symbol indicates that the difference between the corresponding groupand wild-type mice is statistically significant (P<0.05). WT, wild type;p19ko, IL-23p19^(−/−) mice; p40ko, IL-12p40^(−/−) mice.

FIGS. 12A and 12B: Normal T-cell priming yet reduced levels of IL-17production by IL-23p19^(−/−) antigen presenting cells:

FIG. 12A shows in vitro allostimulation experiment of balb/c T-cells incombination with wild-type (black bars) or IL-23p19^(−/−) (white bars)dendritic cells. Naïve CD4⁺ T-cells and CD8⁻/CD11c⁺/MHC-II⁺ cells wereisolated by FACS and incubated in the presence or absence of bacteriallipopeptides (BLP). Proliferation and cytokine levels in thesupernatants were determined after a 5-day incubation period. APC,antigen presenting cells.

FIG. 12B shows In vivo T-cell response. Lymph node cell suspensions fromwild-type (black bars) or IL-23p19^(−/−) mice (white bars) immunizedwith KLH were isolated and restimulated in vitro with 25 μg/ml KLH.Proliferation and IL-17 levels were measured after 5 days in culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

The term “antagonist” is used herein in the broadest sense. An IL-23“antagonist” is a molecule, which partially or fully bocks, inhibits,neutralizes, prevents or interferes with a biological activity of IL-23,regardless of the underlying mechanism. For the purpose of the presentinvention, the biological activity preferably is the ability to induceIL-17 production in activated T cells. Antagonists of IL-23 can beidentified, for example, based upon their ability to inhibit, block, orreverse IL-23 mediated IL-17 production in activated (e.g. memory) Tcell populations. For example a culture of activated T cells can beincubated with IL-23, in the presence and absence of a test compound,and IL-17 level monitored in the cell culture supernatant, e.g. byELISA. If the IL-17 level is lower in the presence of the test compoundthan in its absence, the test compound is an IL-23 antagonist.Alternatively, real-time RT-PCR can be used to monitor IL-17 mRNAexpression in a tissue also expressing IL-23, before and after treatmentwith a test compound. Decrease in IL-17 mRNA level in the presence ofthe test compound indicates that the compound is an IL-23 antagonist.Examples of IL-23 antagonists include, without limitation, neutralizingantibodies against a subunit, e.g. a p40 subunit, of a native sequenceIL-23 polypeptide, immunoadhesins comprising an IL-23 subunit fused toan immunoglobulin constant region sequence, small molecules, antisenseoligonucleotides capable of inhibiting translation and/or transcriptionof a gene encoding a subunit of a native sequence IL-23 polypeptide,decoys, e.g. genetic decoys of the IL-23 gene, etc. Similarly, IL-23antagonist include, without limitation, neutralizing antibodies againsta subunit, e.g. an IL-12Rβ1 or IL-23R subunit, of a native IL-23receptor, immunoadhesins comprising an IL-23 receptor subunit fused toan immunoglobulin constant region sequence, small molecules, antisenseoligonucleotides capable of inhibiting translation and/or transcriptionof a gene encoding a subunit of a native sequence IL-23 receptorpolypeptide, decoys, e.g. genetic decoys of an IL-23 receptor gene, etc.

The term “agonist” is used herein in the broadest sense. An IL-23agonist is any molecule that mimics a biological activity mediated by anative sequence IL-23, regardless of the underlying mechanism. For thepurpose of the present invention, the biological activity preferably isthe ability to induce IL-17 production in activated T cells. Examples ofIL-23 agonists include, without limitation, agonist antibodies against asubunit, e.g. an IL-12Rβ1 or IL-23R subunit, of a native IL-23 receptor,peptides and small organic molecules.

“Antisense oligodeoxynucleotides” or “antisense oligonucleotides” (whichterms are used interchangeably) are defined as nucleic acid moleculesthat can inhibit the transcription and/or translation of target genes ina sequence-specific manner. The term “antisense” refers to the fact thatthe nucleic acid is complementary to the coding (“sense”) geneticsequence of the target gene. Antisense oligonucleotides hybridize in anantiparallel orientation to nascent mRNA through Watson-Crickbase-pairing. By binding the target mRNA template, antisenseoligonucleotides block the successful translation of the encodedprotein. The term specifically includes antisense agents called“ribozymes” that have been designed to induce catalytic cleavage of atarget RNA by addition of a sequence that has natural self-splicingactivity (Warzocha and Wotowiec, “Antisense strategy: biological utilityand prospects in the treatment of hematological malignancies.” Leuk.Lymphoma 24:267-281 [1997]).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including antagonist, e.g. neutralizingantibodies and agonist antibodies), polyclonal antibodies,multi-specific antibodies (e.g., bispecific antibodies), as well asantibody fragments. The monoclonal antibodies specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]). The monoclonalantibodies further include “humanized” antibodies or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); and Reichmann et al., Nature, 332:323-329 (1988).The humanized antibody includes a PRIMATIZED®□ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

As used herein, the term “inflammatory disease” or “inflammatorydisorder” refers to pathological states resulting in inflammation,typically caused by neutrophil chemotaxis. Examples of such disordersinclude inflammatory skin diseases including psoriasis and atopicdermatitis; systemic scleroderma and sclerosis; responses associatedwith inflammatory bowel disease (IBD) (such as Crohn's disease andulcerative colitis); ischemic reperfusion disorders including surgicaltissue reperfusion injury, myocardial ischemic conditions such asmyocardial infarction, cardiac arrest, reperfusion after cardiac surgeryand constriction after percutaneous transluminal coronary angioplasty,stroke, and abdominal aortic aneurysms; cerebral edema secondary tostroke; cranial trauma, hypovolemic shock; asphyxia; adult respiratorydistress syndrome; acute-lung injury; Behcet's Disease; dermatomyositis;polymyositis; multiple sclerosis (MS); dermatitis; meningitis;encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmunediseases such as rheumatoid arthritis (RA), Sjorgen's syndrome,vasculitis; diseases involving leukocyte diapedesis; central nervoussystem (CNS) inflammatory disorder, multiple organ injury syndromesecondary to septicaemia or trauma; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases includingglomerulonephritis; sepsis; sarcoidosis; immunopathologic responses totissue/organ transplantation; inflammations of the lung, includingpleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis,idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; etc. Thepreferred indications include, without limitation, chronic inflammation,autoimmune diabetes, rheumatoid arthritis (RA), rheumatoid spondylitis,gouty arthritis and other arthritic conditions, multiple sclerosis (MS),asthma, systhemic lupus erythrematosus, adult respiratory distresssyndrome, Behcet's disease, psoriasis, chronic pulmonary inflammatorydisease, graft versus host reaction, Crohn's Disease, ulcerativecolitis, inflammatory bowel disease (IBD), Alzheimer's disease, andpyresis, along with any disease or disorder that relates to inflammationand related disorders.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain thedesired effect for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman.

The term “mammal” is used herein to refer to any animal classified as amammal, including, without limitation, humans, domestic and farmanimals, and zoo, sports, or pet animals, such as sheep, dogs, horses,cats, cows, etc. Preferably, the mammal herein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic (including preventative) results. An effectiveamount can be administered in one or more administrations.

B. Modes of Carrying Out the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, 2^(nd) edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology”, 4^(th) edition (D.M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991).

As discussed before, the invention is based on the recognition thatIL-23 induces IL-17 production in activated T cell, in particular memorycells, and that IL-23 antagonists are capable of inhibiting thisprocess. Accordingly, IL-23 antagonists are promising drug candidatesfor the treatment of inflammatory conditions characterized by elevatedlevels of IL-17. Conversely, IL-23 agonists are useful to induceprotective immune response to various infections, includingMycobacterial infections, such as, for example, Mycobacteriumtuberculosis (M. tuberculosis) infection.

1. Screening Assays to Identify IL-23 Antagonists or Agonists

This invention includes screening assays to identify IL-23 antagonists,which find utility in the treatment of inflammatory conditionscharacterized by the presence of elevated levels of IL-17. The inventionfurther includes screening assays to identify IL-23 agonists that findutility in stimulating a protective immune response to infections, suchas infections by Mycobacterium tuberculosis.

Screening assays for antagonist drug candidates may be designed toidentify compounds that bind or complex with IL-23 (including a subunitor other fragment thereof) or with an IL-23 receptor (including asubunit or other fragment thereof), or otherwise interfere with theinteraction of IL-23 with other cellular proteins, thereby interferingwith the production or functioning of IL-23. The screening assaysprovided herein include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Generally, binding assays and activityassays are provided.

Screening assays for antagonist drug candidates may be designed toidentify compounds that bind or complex with IL-23 (including a subunitor other fragment thereof) or with an IL-23 receptor (including asubunit or other fragment thereof), or otherwise interfere with theinteraction of IL-23 with other cellular proteins, thereby interferingwith the production or functioning of IL-23. The screening assaysprovided herein include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Generally, binding assays and activityassays are provided.

The assays can be performed in a variety of formats, including, withoutlimitation, protein-protein binding assays, biochemical screeningassays, immunoassays, and cell-based assays, which are wellcharacterized in the art.

All assays for antagonists and agonists are common in that they call forcontacting the drug candidate with an IL-23 polypeptide, or and IL-23receptor polypeptide, or a fragment of such polypeptides (specificallyincluding IL-23 and IL-23 receptor subunits) under conditions and for atime sufficient to allow these two components to interact. For example,the human IL-23 p19 subunit is a 189 amino acid polypeptide, thesequence of which is available from the EMBL database under AccessionNumber AF301620 (NCBI 605580; GenBank AF301620; Oppmann et al., supra).The sequence of subunit p40 of the IL-23 polypeptide is also known (alsoknown as IL-12 p40 subunit; NCBI 161561). The sequence of IL-12Rβ1, towhich IL-23 binds, is available under Accession Number NCBI 601604. Themaking of antibodies or small molecules binding to such polypeptides iswell within the skill of the ordinary artisan. In binding assays, theinteraction is binding, and the complex formed can be isolated ordetected in the reaction mixture. In a particular embodiment, either theIL-23 or IL-23 receptor polypeptide or the drug candidate is immobilizedon a solid phase, e.g., on a microtiter plate, by covalent ornon-covalent attachments. Non-covalent attachment generally isaccomplished by coating the solid surface with a solution of the IL-23or IL-23 receptor polypeptide and drying. Alternatively, an immobilizedantibody, e.g., a monoclonal antibody, specific for the IL-23polypeptide or the IL-23 receptor polypeptide to be immobilized can beused to anchor it to a solid surface. The assay is performed by addingthe non-immobilized component, which may be labeled by a detectablelabel, to the immobilized component, e.g., the coated surface containingthe anchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

If the candidate compound is a polypeptide which interacts with but doesnot bind to IL-23 or the IL-23 receptor, its interaction with therespective polypeptide can be assayed by methods well known fordetecting protein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fieldsand Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, the other one functioning as the transcription-activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

Compounds that interfere with the interaction of IL-23 and other intra-or extracellular components, in particular IL-17, can be tested asfollows. Usually a reaction mixture is prepared containing IL-23 and theintra- or extracellular component (e.g. IL-17) under conditions and fora time allowing for the interaction of the two products. To test theability of a candidate compound to inhibit the interaction of IL-23 andIL-17, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. Since IL-23 has been shown toinduce IL-17 production, the ability of the test compound to inhibit theIL-23/IL-17 interaction can, for example, be tested by measuring theamount of IL-17 in the absence and presence of the test compound. If theIL-17 amount is lower in the absence of the candidate compound than inits presence, the candidate compound is an IL-23 antagonist by thedefinition of the present invention.

The IL-23 antagonists identified based upon their ability to inhibit theinduction of IL-17 production by IL-23 are drug candidates for thetreatment of inflammatory conditions characterized by the presence ofelevated levels of IL-17.

The IL-23 agonists identified by upon their ability to promote theinduction of IL-17 production by IL-23 are drug candidates for evokingor supporting a protective immune response to infections, such asinfection by Mycobacterium tuberculosis, and, as a result, for thetreatment of infectious diseases, such as tuberculosis.

It is emphasized that the screening assays specifically discussed hereinare for illustration only. A variety of other assays, which can beselected depending on the type of the antagonist candidates screened(e.g. polypeptides, peptides, non-peptide small organic molecules,nucleic acid, etc.) are well know to those skilled in the art and areequally suitable for the purposes of the present invention.

2. Anti-IL-23 and Anti-IL-23 Receptor Antibodies

In a particular embodiment, the IL-23 antagonists or agonists aremonoclonal antibodies to IL-23 (e.g. a subunit of IL-23), includingantibody fragments. In another particular embodiment, the IL-23antagonists and agonists include monoclonal antibodies to an IL-23receptor (e.g. a subunit of an IL-23 receptor). IL-23, including itssubunits, has been discussed hereinabove. The receptor for IL-23 iscomprised of two subunits, IL-12Rβ1, and a more recently discoveredsubunit termed IL-23R (Parham et al., J. Immunol. 168:5699-5798 (2002)).Antibodies to either subunit are specifically within the scope of theinvention. In case of antagonists, antibodies specifically binding theIL-23R subunit are particularly preferred, since they specifically blockthe biological activities mediated by IL-23.

Methods for making monoclonal antibodies are well known in the art.Thus, monoclonal antibodies may be prepared using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975).In a hybridoma method, a mouse, hamster, or other appropriate hostanimal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the IL-23 or IL-23 receptorpolypeptide or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against IL-23or an IL-23 receptor. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.

The anti-IL-23 and anti-IL-23 receptor antibodies of the invention mayfurther be humanized antibodies or human antibodies. Humanized forms ofnon-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Mendez et al. (Nature Genetics 15: 146-156 (1997)) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 (1990)) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires has been described by Waterhouse et al., Nucl. Acids Res.21, 2265-2266 (1993).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). In anotherembodiment, the F(ab′)₂ is formed using the leucine zipper GCN4 topromote assembly of the F(ab′)₂ molecule. According to another approach,Fv, Fab or F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Heteroconjugate antibodies, composed of two covalently joinedantibodies, are also within the scope of the present invention. Suchantibodies have, for example, been proposed to target immune systemcells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment ofHIV infection (PCT application publication Nos. WO 91/00360 and WO92/200373). Heteroconjugate antibodies may be made using any convenientcross-linking methods, using well known, commercially availablecross-linking agents.

For further information concerning the production of monoclonalantibodies see also Goding, J. W., Monoclonal Antibodies: Principles andPractice, 3rd Edition, Academic Press, Inc., London, San Diego, 1996;Liddell and Weeks: Antibody Technology: A Comprehensive Overview, BiosScientific Publishers: Oxford, UK, 1995; Breitling and Dubel:Recombinant Antibodies, John Wiley & Sons, New York, 1999; and PhageDisplay: A Laboratory Manual, Barbas et al., editors, Cold SpringsHarbor Laboratory, Cold Spring Harbor, 2001.

3. Target Diseases

IL-17 has been implicated in various inflammatory diseases, includingrheumatoid arthritis (RA). One of the cardinal features of RA is erosionof periarticular bone. Osteoclasts play a key role in bone resorptionbut the mechanisms by which osteoclasts are formed from progenitor cellsis not fully understood. Recently, Kotake, et al. (J. Clin. Invest.103:1345 (1999)) reported that Interleukin 17 (IL-17) could induce theformation of osteoclast-like cells in cocultures of mouse hemopoieticcells and primary osteoblasts. This IL-17 induced osteoclastogenesis wasshown to be inhibited by indomethacin, a selective inhibitor ofcyclooxygenas-2 (COX-2). The synovial fluids from RA patients were foundto contain significantly higher levels of IL-17 as compared toosteoarthritis (OA) patients. In addition, using immunostaining,IL-17-positive mononuclear cells were detected in the synovial tissuesof RA patients and not in tissue from OA patients. These findings havebeen interpreted to indicate that IL-17 may contribute to bone erosionand joint damage in RA and may therefore, be a target for inhibition.

Behcet's disease patients have also been shown strinkingly elevatedserum levels of IL-17 compared to healthy subjects. Hamzaoui et al.,Scand. J. Rheumatol. 31(4):205-10 (2002).

Elevated levels of IL-17 have been found within asthmatic airways, andit has been suggested that IL-17 might amplify inflammatory responsesthrough the release of other proinflammatory mediators, such asalpha-chemokines. Molet et al., J. Allergy Clin. Immunol. 108(3):430-8(2001); and Wong et al., Clin. Exp. Immunol. 125(2):177-83 (2001).

Elevated levels of IL-17 have been reported for patients with systhemiclupus erythrematosus. Wong et al., Lupus 9(8):589-93 (2000).

IL-17 has been described to play a role in psoriasis. Homey et al., J.Immunol. 164(12):6621-32 (2000).

It has been reported that IL-17 mRNA is augmented in blood and CSFmononuclear cells in multipe sclerosis. Matusevicius et al., Mult.Scler. 5(2):101-4 (1999).

Based on these and numerous similar reports, IL-23 antagonists, whichinhibit the ability of IL-23 to induce IL-17 production, and therebylower IL-17 levels, are valuable candidates for the treatment of avariety of (chronic) inflammatory conditions and diseases. Examples ofsuch conditions and diseases include, without limitation: chronicinflammation, autoimmune diabetes, rheumatoid arthritis (RA), rheumatoidspondylitis, gouty arthritis and other arthritic conditions, multiplesclerosis (MS), asthma, systhemic lupus erythrematosus, adultrespiratory distress syndrome, Behcet's disease, psoriasis, chronicpulmonary inflammatory disease, graft versus host reaction, Crohn'sDisease, ulcerative colitis, inflammatory bowel disease (IBD),Alzheimer's disease, and pyresis.

IL-17 is known to play an important role in the generation of aprotective response to certain infectious diseases, such as tuberculosisby promoting IFN-γ production and thereby inducing a cell-mediatedimmune response. Accordingly, IL-23 agonists, including agonistantibodies, find utility in inducing a cell-mediated immune response tovarious infections, such as tuberculosis causes by Mycobacteriumtuberculosis, and are promising drug candidates for treating thisinfectious disease which kills more than three million people worldwideevery year.

4. Pharmaceutical Compositions

Antibodies specifically binding IL-23 or an IL-23 receptor, as well asother IL-23 antagonist or agonist molecules identified by the screeningassays disclosed hereinbefore, can be administered for the treatment ofvarious disorders, in particular inflammatory diseases or diseasesbenefiting from the induction of cell-mediated immune response, in theform of pharmaceutical compositions.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended, or may be formulated separately, andadministered concurrently or consecutively, in any order.

For example, the IL-23 antagonists of the present invention may beadministered in combination with anti-inflammatory agents and otheractive compounds currently in use for the treatment of the targetdiseases and conditions. Such compounds include corticosteroids;non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin,ibuprofen, and COX-2 inhibitors, e.g. Celebrex® and Vioxx®;disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate,leflunomide, sulfasalazine, azathioprine, cyclosporine,hydroxychloroquine, and D-penicillamine; and biological responsemodifiers (BRMs), such as TNF and IL-1 inhibitors.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

Example 1 Interleukin-23 (IL-23) Promotes a Distinct CD4 T CellActivation State Characterized by the Production of Interleukin-17(IL-17)

Although clearly produced by activated T cells, previous reports havenot provided clear classification of IL-17 within the paradigm of Th1and Th2 polarized cytokine profiles. The purpose of the initialexperiments described in this Example was to examine the possibilitythat IL-17 is expressed in response to signals distinct from thoseassociated with the Th1 or Th2 response.

Experimental Procedures

Cell Culture

Single cell suspensions of spleen were prepared from C57/BL-6 mice, andmononuclear cells were isolated from suspended splenocytes by densitygradient centrifugation. 2×10⁶ cells/ml were cultured with IL-2 (100units/ml) in the presence or absence of various stimuli (for timesindicated in the figure legends), following which the cells werecollected and analyzed for IL-17 using ELISA (R&D Systems, Minneapolis,Minn.). Dendritic cells were derived from macrophages (obtained asadherent population from splenocyte suspension) by treating mactophageswith rGM-CSF (2 ng/ml) and rIL-4 (1000 units/ml) for 4 days, washing andre-activating using LPS (0.5 μg/ml). Memory and naive T cells wereisolated by staining mononuclear cells isolated from single cellsuspension of murine splenocytes with CyC-CD4+PE-CD44 orCyC-CD4+PE-CD62L and sorting for CD4⁺ cells that were eitherCD44^(high)/CD62L^(low) for memory phenotype, or CD44^(low)/CD62^(high)for naive phenotype.

In Vitro Induction of T Cell Differentiation

CD4⁺ cells were purified from spleen of wild type C57/BL6 mice usinganti-CD4 magnetic beads (Miltenyi Biotech). Purified T cells (2×10⁶cells/ml) were activated for 3 days by plating on plates coated with 5μg/ml anti-CD3 and 1 g/ml anti-CD28 antibodies. The cultures weresupplemented with IL-2 and treated with IL-12 (20 mM)+anti-IL4 (0.5μg/ml) (for Th1 differentiation), or IL-23 (10 nM) (for IL-17production). Following initial activation, the cell cultures were washedextensively and re-stimulated with anti-CD3 (1 μg/ml) for another 24 h,following which the cell supernatants were analyzed for various secretedcytokines using ELISA.

IL-12p40 Antibody Inhibition of IL-17 Induction

Anti-IL-12 antibody (R&D Systems, cat. no. AF-419-NA) or an unrelatedcontrol antibody (anti-FGF-8b (R&D Systems, cat. no. AF-423-NA) werepre-incubated with IL-23 (100 ng/ml) or conditioned media ofLPS-stimulated dendritic cells (10% v/v) for 1 h at 37° C. and thenincubated for another 5-6 days with mononuclear cells isolated frommouse spleen (2×10⁶ cells/ml). Supernatants were collected and levels ofIL-17 measured using ELISA.

Purification of IL-23-Murine IL-23

IL-23 component was produced by co-expression of carboxyl-terminalHis-tagged p19 and FLAG0tagged p40 in human embryonic kidney cells (293cells), and secreted protein was purified by nickel affinity resin.Endotoxin levels were undetectable at less than 0.2 endotoxin units perμg.

Results

First, the ability of various microbial products to stimulate theproduction of IL-17 was examined. Increased IL-17 has recently beenobserved by Infante-Duarte et. al., J Immunol 165, 6107 (2000), inresponse to microbial lipopeptides from a Lyme disease causingspirochete, B. burgdorferi. Spleen cell cultures in the presence ofvarious microbial peptides including LPS (gram-negative bacteria), LTA(gram positive bacteria) or LBP (bacterial lipopeptide) resulted in theproduction of IL-17 (FIG. 1). Neither purified T cells alone, norpurified macrophages themselves produced IL-17. Purified T cells, uponreceptor cross-linking using plate-bound anti-CD3 and treatment withsupernatants from activated macrophages/dendritic cells producedincreased IL-17, indicating the presence of an unidentified factor(s)released by these cells that acts on T cells to promote IL-17production.

In profiling the expression of candidate molecules that might beresponsible for this IL-17 promoting activity, a 100-1000 fold increasedmRNA expression of the IL-23 (B. Oppmann et al., Immunity 13, 715(2000)) components p19 and p40 was observed in activated dendritic cellsusing real-time RT-PCR (not shown), hence, the effect of IL-23 wasexamined.

Murine IL-23 component was produced by co-expression of carboxylterminal His-tagged p19 and Flag-tagged p40 in human embryonic kidneycells (293 cells) and secreted protein was purified by nickel affinityresin. Endotoxin levels were undetectable at less than 0.2 EU per μg.Spleen cell cultures were incubated in presence of IL-2 (100 U/ml) andConA (2.5 μg/ml) under Th1-inducing conditions (IL-12+ anti-IL-4),Th2-inducing conditions (IL-4+ anti-IFN-γ), or purified IL-23 (100ng/ml) for 3-4 days, following which, the cultures were washed andre-stimulated with ConA for another 24 hours. Levels of variouscytokines were measured using ELISA. The levels less than the lowestdilution of the standard curve range of ELISA kit were recorded as ‘notdetectable (N.D.)’. The results below are representative of threeexperiments performed independently.

Spleen cells, cultured under IL-12-stimulated Th1-inducing conditionsresulted in marginal IL-17 production, whereas under Th2-inducingconditions there was no increased production of IL-17 over controls. Theresults are shown in the following Table 1.

TABLE 1 Control IL-12 IL-4 IL-23 IL-17 N.D 58 ± 82 64 ± 91 1191 ± 569 IL-4 50 ± 26 396 ± 17  3259 ± 118  101 ± 100 IFN-γ 341 ± 0  2757 ± 1016489 ± 502 580 ± 813 GM-CSF N.D. 46 ± 13 365 ± 516 882 ± 169 TNF-α N.D174 ± 40  214 ± 314 205 ± 85 

Presence of IL-23 in cultures resulted in high level IL-17 production,in a dose-dependent manner (FIG. 2). IL-23 also resulted in higherlevels of GM-CSF than observed under Th1-inducing conditions. Incontrast, IFN-γ levels were significantly lower than those obtainedunder Th1-inducing conditions. TNF-α levels were similar to Th1conditions. IL-12p40 alone did not result in any IL-17 production (datanot shown). IL-23 promoted elevated levels of IL-17 mRNA (FIG. 2B).IL-17 mRNA levels were increased several hundred-fold within 6 h ofIL-23 exposure and remained elevated in the continued presence of IL-23.This effect was no inhibited by the presence of an antibody againstIL-17, suggesting that the IL-17 itself was not contributing to thisprocess (not shown). In addition, mRNA for IL-17F, a recently identifiedIL-17 family member, was also found to be upregulated in response toIL-23 (FIG. 2C).

IL-23 has been reported to promote the proliferation of memory but notnaïve T cells (D. M. Frucht, supra. Therefore, the effect of IL-23 onIL-17 production from naïve versus memory T cell populations wasexamined. Purified CD4⁺ T cells were isolated from splenocytes byfluorescence activated cell sorting (FACS). The memory cell populationwas selected as CD4⁺CD44^(high) (R. C. Budd et al., J Immunol 138, 3120(1987)), or CD4⁺CD62L^(low) (T. M. Jung, W. M. Gallatin, I. L. Weissman,M. O. Dailey, J Immunol 141, 4110 (1988)), and naïve cell population wasselected as CD4⁺CD44^(low) or CD4⁺CD62L^(high). As seen in FIG. 3, IL-23stimulated IL-17 production only in memory cell population (CD44^(high)and CD62L^(low)) and not in naïve cells (CD44^(low) or CD62L^(high)).

The IL-23-mediated IL-17 production was completely blocked in thepresence of a neutralizing IL-12 antibody that interacts with the p40subunit shared with IL-23 (FIG. 4A, left panel). This effect was not dueto ligation of Fc receptors on antigen presenting cells as there was nochange in IL-17 production in the presence of unrelated antibody. Thisantibody also inhibited >50 percent the induction of IL-17 productionobserved in response to conditioned media from LPS stimulated dendriticcells (FIG. 4A, right panel). A marked reduction, but not abrogation, ofIL-17 production was seen in response to ConA stimulation from spleencell cultures of mice lacking IL-12p40 component (strain:B6.129S1-IL12b^(tm1Jm)) as compared to wild type mice or mice lackingIL-12p35 component (strain: B6.129S1-IL12a^(tm1Jm)) (FIG. 4B).

In order to examine the role of IL-12 in IL-17 production, increasingamounts (0.001-1 nM) of murine IL-12 were added to IL-23 (1 nM)containing cultures. As seen in FIG. 5A, IL-12 decreased IL-17 levels ina dose dependent manner.

Additionally, splenocytes from mice lacking IL-12 receptor beta chain 2(IL-12Rβ2) (Wu et al., J Immunol 165, 6221 (2000)), the specificreceptor component of IL-12 (A. O. Chua, V. L. Wilkinson, D. H. Presky,U. Gubler, J Immunol 155, 4286 (1995)), were treated with purifiedIL-23. Splenocytes from IL-12Rβ2^(−/−) mice responded to IL-23 stimulusby increasing IL-17 production over the un-stimulated control (FIG. 5B)without affecting IFN-γ levels. Surprisingly, the background levels ofIL-17 in these mice were more than 10-fold as compared to wild-typemice, suggesting a possible negative regulation by IL-12 ofIL-23-induced IL-17 production. However, in contrast to IL-12Rβ2knockout mice, we did not observe increased IL-17 in spleen culturesfrom IL-12p35 knockout mice. The reasons for this difference are notknown, but could relate to alteration in IL-12p40 function in theabsence of p35, or differences in genetic background or pathogenexposure.

Discussion

Taken together, these data suggest a role for IL-23 in the promotion ofa distinct T cell activation state that expresses IL-17 as an effectorcytokine. The Th1 and Th2 paradigms have been described as promotingcell mediated versus humoral immune responses. These responses provideimportant defense for intracellular and extracellular pathogensrespectively, and defects in either of these responses are associatedwith increased susceptibility to specific pathogens. In contrast, IL-23may serve to promote an adaptive immune response to pathogens that ischaracterized by a heavy reliance on cells thought to function primarilyas mediators of the innate immune response. IL-17, as a principleeffector cytokine of this response, is able to promote the more rapidrecruitment of monocytes and neutrophils through induced chemokineproduction. In addition, the high level GM-CSF production observed inresponse to IL-23 supports the production of additional myeloid cells.This is further augmented by G-CSF production from localIL-17-stimulated stromal cells. The character of this adaptive responseis, however, not an exclusive reliance on phagocytic cells of themyeloid lineage response as IL-17 is known to promote the induction ofICAM by IL-17 thereby providing important co-stimulation of further Tcells responses.

Recently, several studies have pointed out significant differencesbetween mice deficient in p35 and mice deficient in p40 (Decken et al.,Infect Immun. 66:4994-5000 (2002); Cooper et al., J. Immunol.168:1322-1327 (2002); Elkins et al., Infection & Immunity 70:1936-1948;Holscher et al., J. Immunol. 167:6957-6966 (2001)). These studies sharethe observation that loss of p40 is generally more deleterious than lossof p35 in the immune-mediated clearance of a variety of model organisms.

The association of IL-17 expression with a number of seriousinflammatory diseases suggests that IL-23 antagonists may be promisingdrug candidates in the treatment of such diseases.

Example 2 Interleukin-23 (IL-23) Deficient Mice

To further investigate the relationship between IL-23 and IL-17 in vivo,the phenotype of IL-23 deficient mice was compared to that of IL-17deficient animals.

Experimental Procedures

Mice:

All mice were housed under specific pathogen free conditions.IL-12p40^(−/−) mice were obtained from the Jackson laboratory (BarHarbor, Mass.), and C57BL/6 were obtained from Charles Riverlaboratories (San Diego, Calif.).

Reagents:

Unless otherwise indicated, reagents were purchased from the followingsuppliers: Antibodies and ELISA reagents were obtained from BDPharmingen (San Diego, Calif.), cytokines from R&D systems (Minneapolis,Minn.), TNP-coupled antigens from Biosearch Technologies (Novato,Calif.) and tissue culture reagents from Invitrogen (Carlsbad, Calif.).

Generation of IL23p19 Deficient Mice.

Genomic DNA encompassing the murine IL23p19 locus was isolated fromclone 198a3 of a genomic BAC library by Genome Systems (Incyte Genomics,Palo Alto, Calif.). A targeting vector designed to replace the entireIL23p19 coding region with an EGFP reporter gene was constructed fromthe following DNA fragments using standard molecular cloning techniques:a thymidine kinase selection cassette; a 5′ homology arm of 5403 basepairs defined by endogenous SacII and BglII sites on the distal andproximal ends, respectively; an EGFP expression cassette excised frompEGFP-1 (BD Clontech, Palo Alto, Calif.) using BamHI (5′-end) and AflIII(3′-end); a PGK-neo resistance cassette; and a 1203 bp short arm definedby an endogenous XhoI site at the proximal end and the primer5′-GCTTGGTGGCCCACCTATGAT-3′ (SEQ ID NO: 1) at the distal end (FIG. 6A).This construct was electroporated into 129/SvEv embryonic stem (ES)cells (Huang et al., Science 259:1742 (1993)) and homologousrecombination occurred in 9 out of 600 clones following selection withG418 and Gancyclovir. To verify correct targeting of the locus, genomicDNA from ES cells and animals was analyzed by southern blot. Digestionwith BamHI followed by hybridization of membranes with probe 1 (a 831 bpgenomic DNA fragment obtained by PCR with oligos5′-AGACCCTCAAAGTTCATGAC-3′ (sense) (SEQ ID NO: 2) and5′-CTGACGGCGCTTTCTCTACC-3′ (antisense) (SEQ ID NO: 3)) yielded a 7027 bpfragment for the wild-type allele and an 11788 bp fragment for thecorrectly targeted mutant allele. Similarly, digestion of genomic DNAwith EcoRI followed by hybridization of membranes with probe 2 (a 390 bpgenomic DNA fragment obtained by PCR with oligos5′-TTTTGCCAGTGGGATACACC-3′ (sense) (SEQ ID NO: 4) and5′-AACTGCTGGGGCTGTTACAC-3′ (antisense) (SEQ ID NO: 5)) yielded a 9197 bpfragment for the wild-type allele and an 6211 bp fragment for thecorrectly targeted mutant allele. Two ES cell clones (1c5 and 3h6) wereinjected into blastocysts, and chimeric animals that transmitted themutant allele in their germline were obtained. For routine genotyping,we used a PCR-based method with a common antisense primer(5′-GCCTGGGCTCACTTTTTCTG-3′) (SEQ ID NO: 6), and wild-type specific(5′-GCGTGAAGGGCAAGGACACC-3′) (SEQ ID NO: 7) and knockout-specific(5′-AGGGGGAGGATTGGGAAGAC-3′ (SEQ ID NO: 8)) sense primers. Thisprimer-triplet amplifies a 210 bp fragment for the wild-type allele anda 289 bp fragment for the mutant allele. PCR was carried out in aRobocycler (Stratagene, La Jolla, Calif.), using the followingconditions: 1 cycle of 94° C., 60″; 35 cycles of 94° C., 30″, 58° C.,30″, 72° C., 60″; 1 cycle of 72° C., 7″.

FACS Analysis of Blood Cell Subsets:

Spleens, thymi, and lymph nodes were isolated from 6-8 week old mice,and single cell suspensions were prepared by standard methods.Peripheral blood was obtained by cardiac puncture and treated with EDTAto prevent coagulation, and erythrocytes were lysed using ACK lysingbuffer (Biosource, Camarillo, Calif.). All cells were incubated for 30minutes on ice in Hanks balanced salt solution (HBSS) supplemented with2% heat inactivated bovine calf serum. Cells were then stained in thesame buffer with 1 μg per million cells of various antibodies coupled tophycoerythrin, biotin or Cychrome™. Where biotinylated antibodies wereused, streptavidin-coupled PE-TR conjugate (Caltag, Burlingame, Calif.)was used for detection. After two washes with the same buffer,fluorescence was detected using an Epics-XL flow cytometry system(Beckman Coulter Inc., Fullerton, Calif.).

Stimulation of Allotypic T-Cells:

CD4 and CD62L double positive T-cells were isolated from the spleens of6-8 week old balb/c mice by a two-step isolation protocol. First,T-cells were depleted of other cell types by a negative magneticselection (Miltenyi, Auburn, Calif.). These cells were then labeled withantibodies against CD4 and CD62L and sorted by FACS on a MoFlo sorter(DakoCytomation, Fort Collins, Colo.). Dendritic cells from wild type orIL-23p19^(−/−) mice, both in the C57BL/6 background, were also isolatedby a two-step protocol. CD11c positive splenocytes were positiveselected by magnetic separation (Miltenyi, Auburn, Calif.) prior tolabeling with antibodies against CD11c, MHC class II, and CD8.CD11c⁺/MHC-II⁺/CD8⁻ cells were then sorted by FACS, again using a MoFlosorter. All populations used in the experiment were at least 98% pure.To elicit allostimulatory responses, 10⁴ dendritic cells and 10⁵ T-cellswere incubated in a total of 200 μl of IMDM supplemented withpenicillin-streptomycin and 10% heat inactivated bovine calf serum(Hyclone, Logan, Utah) in duplicates. In some cases, 100 ng/ml bacteriallipopeptides was added to stimulate cytokine production by dendriticcells. After 5 days of incubation, 120 μl of supernatant were removedfor cytokine measurement by ELISA, and replaced with fresh mediumcontaining 1 μCi ³H-thymidine per well. Thymidine incorporation wasdetermined 16 hours later using a Top Count liquid scintillation counteraccording to the manufacturers instructions (Packard Instruments,Meriden, Conn.).

In Vivo T-Cell Differentiation:

Four male and four female mice per group were immunized into the lefthind footpad with 75 μg of keyhole limpet hemocyanin (KLH) (Sigma, St.Louis, Mo.) in 30 μl of a 1:1 emulsion of CFA (BD Biosciences, SanDiego, Calif.) and PBS. Draining inguinal and popliteal lymph nodes wereharvested 5 days later and restimulated in IMDM supplemented withpenicillin-streptomycin, 10% heat inactivated bovine calf serum(Hyclone, Logan, Utah), and 25 μg/ml KLH. For proliferation assay, 5*10⁵cells were seeded in 200 μl in triplicates in 96 well plates and allowedto proliferate for 112 hours with addition of 1 μCi ³H-thymidine perwell during the last 18 hours of the incubation period. Thymidineincorporation was determined using a Top Count liquid scintillationcounter according to the manufacturers instructions (PackardInstruments, Meriden, Conn.). For cytokine secretion, 2.5*10⁶ cells wereincubated in 1 ml in 48 well plates, and supernatants were harvestedafter 72 hours. Cytokine secretion was determined by ELISA. The datapresented is one representative out of three total experiments.

Delayed Type Hypersensitivity Responses:

6 mice per group were subcutaneously injected with 200 μg of methylatedbovine serum albumin (mBSA) (Sigma, St. Louis, Mo.) at three sites inthe abdomen in a combined total of 200 μl of a 1:1 emulsion of CFA (BDBiosciences, San Diego, Calif.) and PBS. On day 8 followingimmunization, the mice were challenged by injection of 20 μl of 5 mg/mlmBSA in PBS into one rear footpad, while the other rear footpad received20 μl of PBS. Measurements of footpad swelling were taken at 18, 42, 66hours after challenge, using a series 7 spring-loaded caliper (Mitutoyo,City of Industry, Calif.). The magnitude of the DTH responses wasdetermined from differences in footpad thickness between the antigen-and PBS-injected footpads.

T-Dependent Humoral Responses and Immunoglobulin Analysis:

For the measurement of total immunoglobulin levels, serum was obtainedfrom 8 male and 8 female, 6-9 week old, unimmunized mice of eithergenotype. Total immunoglobulin isotype levels were measured by Luminexbead assay (Upstate, Lake Placid, N.Y.). To assess the OVA specifichumoral immune response, groups of 7 mice per genotype (4 males and 3females) were immunized with OVA in CFA on day 0 and received boosterimmunizations of the same antigen in incomplete Freund's adjuvant (IFA)(Sigma, St. Louis, Mo.) on days 21 and 42. For serum analysis, blood wasobtained by retro-orbital bleeding before immunization and on days 14,28, and 49 after immunization. OVA specific immunoglobulin isotypes weredetected by ELISA, using OVA as a capture agent and isotype specificsecondary antibodies for detection. In order to be in the linear rangeof the ELISA, serum samples were diluted as follows: 1:3125000 for IgG1,1:25000 for IgG2a, 1:625000 for IgG2b, and 1:1000 for IgG3, IgM, IgA andIgE. A dilution series of a serum obtained from an OVA-immunized mousefrom a previous experiment was used as a standard, since purified, OVAspecific isotypes are not commercially available. Results are expressedas arbitrary units, where the average of the wild-type group in the lastbleed was set as 100. To assess the contribution of memory T-cells tothe humoral response, groups of 5-6 mice of either genotype wereimmunized with OVA in CFA on day 0 and received a booster immunizationof TNP₁₁-OVA in IFA on day 21. For serum analysis, blood was obtained byretro-orbital bleeding before immunization and on days 14 and 28 afterimmunization. TNP specific immunoglobulin isotypes were detected byELISA, using TNP₂₈-BSA as a capture agent and isotype specific secondaryantibodies for detection. For TNP-specific IgG1, a commerciallyavailable standard was used. For TNP-specific IgG2a, a dilution seriesof a serum obtained from a TNP immunized mouse from a previousexperiment was used, and results were calculated as described above. Thesample dilutions were 1:31250 for IgG1 and 1:1250 for IgG2a.

T-Independent Humoral Responses:

Groups of 6 mice per genotype were immunized intraperitoneally with 50μg TNP₁-LPS or 100 μg TNP₂₀-AECM-Ficoll in PBS. Serum was harvested 10days later, and TNP-specific IgM was analyzed by ELISA, using TNP₂₈-BSAas a capture agent and an IgM specific secondary antibody for detection.A TNP specific IgM antibody was used as a standard for the ELISA. Thesample dilutions were 1:1280 for Ficoll and 1:5120 for LPS.

Results

Deletion of the IL-23p19 Gene.

To determine the non-redundant in vivo effects of IL-23, mice weregenerated that are deficient in IL-23 but competent to produce IL-12. Atargeting vector was constructed in which the entire coding region ofp19, consisting of 4 exons, is replaced by an enhanced GFP (eGFP)reporter gene, and a neomycin resistance cassette (FIG. 6). Germlinetransmission was obtained from two correctly targeted ES cell clones,1c5 and 3h6, and the mutation was backcrossed into the C57BL/6background using speed congenics with 3 markers per chromosome. Based onthis analysis, only those mice were selected in which the geneticcontamination from the 129 background was less than 5% for experiments.The pattern of eGFP expression was comparable to that of endogenous p19mRNA (data not shown).

IL-23p19^(−/−) Mice have No Overt Phenotype.

As expected from the phenotype of IL-23/IL-12 double deficientIL-12p40^(−/−) mice, IL-23p19^(−/−) animals did not display any overtphenotype and were born at mendelian frequencies. No abnormalities inorgans were found upon histopathological examination, and furtheranalysis of clinical chemistry and hematology parameters did not revealdifferences between wild type and knockout animals. Furthermore,IL-23p19^(−/−) mice were normal in size and weight, and both sexes werefully fertile. Flow cytometric analysis of thymocytes, splenocytes, andperipheral blood leukocytes with various cell surface markers did notindicate any major differences between wild-type and IL23p19^(−/−)animals (Table 2). Because IL-23 is known to act on memory T-cells, wethe ratio of memory (CD44^(high) CD62L⁻) versus naïve (CD62L⁺) cells ofeach subset was determined, but no difference was found betweenwild-type and IL-23p19^(−/−) mice. In the entire analysis, the onlynoticeable difference between the two genotypes consists in a slightskewing of the dendritic cell subpopulations towards a CD8⁺ phenotype.While the effect was minor, it reached statistical significance due tothe tightness of the data, and could be compatible with recentobservations that IL-23 has effects on antigen presenting cells. Insummary, IL-23 does not appear to be required for normal development,and the introduction of an eGFP cassette does not have a toxic effect onany cell type tested.

TABLE 2 wild-type knockout P (diff.) Thymus CD4+  5.7 +/− 0.5  5.5 +/−0.0 0.504 CD8+  3.3 +/− 0.1  3.1 +/− 0.3 0.397 DN 25.0 +/− 4.2 17.0 +/−8.0 0.202 DP 65.9 +/− 3.7 74.3 +/− 8.0 0.174 Spleen CD4+ 24.3 +/− 0.822.5 +/− 2.7 0.342 % naïve 69.0 +/− 1.3 67.5 +/− 2.1 0.090 % memory 29.1+/− 1.2 31.0 +/− 1.9 0.029 CD8+ 15.2 +/− 1.2 12.3 +/− 2.0 0.101 % naïve64.1 +/− 5.4 67.0 +/− 2.8 0.199 % memory 18.1 +/− 1.8 18.3 +/− 1.4 0.084I-A(b)+/CD11c+  2.0 +/− 0.2  2.2 +/− 0.2 0.041 % CD8+ 12.8 +/− 0.9 16.3+/− 1.7 0.000 % CD8− 87.2 +/− 0.9 83.6 +/− 1.8 0.000 CD19+ 52.4 +/− 2.055.2 +/− 6.5 0.512 B220+ 52.0 +/− 2.0 55.5 +/− 5.3 0.360 NK1.1+  3.2 +/−0.1  2.8 +/− 0.1 0.055 Peripheral blood CD3+ 47.9 +/− 2.6 44.9 +/− 3.60.053 CD4+ 28.2 +/− 2.3 26.9 +/− 2.5 0.270 CD8+ 16.5 +/− 0.8 15.6 +/−1.7 0.150 CD19+ 43.2 +/− 3.2 45.2 +/− 3.6 0.215 B220+ 44.9 +/− 3.5 46.4+/− 4.8 0.466 DX5+  9.9 +/− 3.0  9.7 +/− 5.0 0.929 CD16+  8.0 +/− 0.9 8.6 +/− 1.5 0.302 I-A(b)+ 44.0 +/− 1.9 45.4 +/− 4.9 0.428

Humoral Immune Responses in IL-23p19^(−/−) Mice.

To determine the role of IL-23 in the generation of a humoral immuneresponse, first total immunoglobulin levels of all isotypes weremeasured in serum of 16 mice of either genotype. There was nostatistically significant difference between wild type andIL-23p19^(−/−) mice (FIG. 7), indicating that the IL-23 is notcritically required for the maintenance of normal immunoglobulin levels.Next, we tested whether IL-23 is involved in the generation of aT-dependent humoral response against a protein antigen delivered inadjuvant. To this end, groups of 7 mice were immunized, each withOvalbumin (OVA), and assessed OVA-specific immunoglobulin isotypes inpreserum (all negative, data not shown), and after each of twoconsecutive immunizations (FIG. 8). After primary immunization, none ofthe groups differed from each other significantly for OVA specific IgG1,IgG2b, IgG3, and IgE. However, significantly reduced levels of OVAspecific IgG2a and IgA in IL-23p19^(−/−) and IL-12p40^(−/−) animals wereobserved after primary immunization. As expected, the levels of allisotypes were increased dramatically after the second immunization. Atthis point, both IL-23p19^(−/−) and IL-12p40^(−/−) mice displayed markedreduction of all isotypes tested. The difference between these twogenotypes was generally not significant, indicating that endogenousIL-12 does not play a major role in the humoral response in the absenceof IL-23.

Because humoral immune responses depend of the proper function of both Band T cells, we next sought to determine by what mechanism IL-23 exertsits stimulatory effects. To test whether B-cell function is directlyaffected by the lack of IL-23, we tested the ability of IL-23 deficientmice to mount B-cell responses against T-independent (TI) antigens. TheTI-1 antigen trinitrophenyl- (TNP-) LPS leads to B-cell activation viaCD14 and TLR4, while the TI-2 antigen TNP-Ficoll activates B-cellsthrough clustering of surface B-cell receptors. IL-23p19^(−/−) micemounted normal B-cell responses to both types of antigens (FIG. 9),indicating that IL-23 does not play a role in T-independent B-cellresponses. Furthermore, B-cells from IL-23p19^(−/−) mice proliferatednormally in vitro in response to LPS, anti-IgM, and anti-CD40 andunderwent normal isotype switching in response to IL-4 (not shown).IL-23 stimulation of B-cells did not lead to increased proliferation orisotype switching (not shown), and thus we conclude that IL-23 does notdirectly affect B-cell function.

Because the humoral immune response was mainly compromised at the stageof the secondary immunization, and because B-cell function appearednormal in IL-23p19^(−/−) mice, we hypothesized that inefficientre-activation of antigen specific helper T-cells might cause thephenotype. To address this question more directly, we immunized groupsof 5-6 mice with OVA on day 0, followed by a secondary immunization withTNP conjugated OVA on day 14. By using this immunization regimen, memoryT-cells specific for OVA are re-activated by the secondary immunization,but a novel set of B-cells with specificity for TNP is activated at thesecondary time point only. Therefore, the OVA specific memory B-cellsubset does not contribute to the formation of TNP-specificimmunoglobulins. Seven days after the booster, we tested for TNPspecific IgG1 and IgG2a in the serum, and found both isotypes to besignificantly reduced in IL-23p19^(−/−) mice (FIGS. 10A and 10B). Thisresult further underlines the importance of IL-23 in T-dependent B-cellresponses.

Delayed Type Hypersensitivity (DTH) Responses in IL-23p19^(−/−) Mice.

To further investigate the function of memory CD4⁺ cells inIL-23p19^(−/−) mice, the ability of these animals to mount DTH responseswas evaluated. DTH responses are strongly T-cell dependent and werereported to be defective in IL-12p40^(−/−) mice, but appear to be normalin mice lacking IL-12p35, suggesting that they might be mediated byIL-23. To address this question, we sensitized groups of 6 wild-type,IL-23p19^(−/−), and IL-12p40^(−/−) animals each with methylated BSA(mBSA) in complete Freund's adjuvant (CFA) and elicited DTH responses 7days later by injection of mBSA into footpads. To control fornonspecific swelling, we also challenged a group of wild-type mice thathad not been sensitized. Specific footpad swelling was measured 18, 42,and 66 hours after the challenge and found to be inhibited to a similardegree in both IL-12p40^(−/−) and IL-23p19^(−/−) mice compared towild-type mice (FIG. 11). The kinetics was also similar, with bothIL-12p40^(−/−) and IL-23p19^(−/−) mice showing strongly reduced swellingat the 42 and 66 but not at the 18 hour time point. Therefore, IL-23 isa principal mediator of DTH responses, and lack of IL-23 leads toinefficient responses by memory CD4⁺ T-cells.

Capacity of IL-23p19^(−/−) Dendritic Cells to Stimulate T-Cells.

To rule out the possibility that the defects observed in IL-23p19^(−/−)mice are due to inefficient T-cell priming by IL-23 deficient antigenpresenting cells, we next investigated the potential of IL-23p19^(−/−)DC to stimulate allotypic naïve CD4⁺ T-cells isolated from the spleensof balb/c mice. In the absence of DC, these T-cells did not proliferatenor secrete appreciable amounts of cytokines (FIG. 12A). Addition of DCof either genotype resulted in robust proliferation and production ofIL-2 in both genotypes. Since we have shown previously that IL-23 is apotent inducer of IL-17, we next induced IL-23 production by DC usingbacterial lipopeptides, a potent Toll-like receptor- (TLR-) 2 agonistand inducer of IL-23 production. Under these conditions, wt DC potentlyinduced IL-17 production by the T-cells (FIG. 12A, bottom panel), whileT-cells stimulated with IL-23p19^(−/−) DC produced significantly lessIL-17. To confirm these observations in a more physiological setting, wenext elicited T-cell responses in vivo by immunizing groups of 8 micewith Keyhole-limpet hemocyanin (KLH) in complete Freund's adjuvant(CFA). Draining lymph node cells (LNC) were harvested 5 days later andre-stimulated with KLH in vitro. Again, we observed that LNC harvestedfrom IL-23p19^(−/−) mice produced significantly less IL-17 (FIG. 12B,bottom panel). LNC proliferation was comparable in both genotypes (FIG.12B, top panel), indicating that both wt and IL-23p19^(−/−) mice mountedrobust T-cell responses against the antigen. Thus, IL-23 deficiency doesnot grossly impair the stimulatory potential of dendritic cells, butresults in attenuated IL-17 production by T-cells.

Discussion

Using IL-23p19 deficient mice, the non-redundant in vivo functions ofIL-23 were assessed, and found that IL-23 deficiency results incompromised T-cell dependent immune responses, such as humoral immuneresponses and DTH reactions.

Profoundly reduced humoral immune responses were observed inIL-23p19^(−/−) mice, affecting all immunoglobulin isotypes. In parallel,responses of IL-12p40^(−/−) mice were inhibited to a similar or slightlyhigher degree. Our results support the conclusion that IL-23 isabsolutely required for an efficient humoral response, while it remainsto be determined, through the use of IL-12p35^(−/−) mice, whether IL-23is sufficient for normal humoral responses in the absence of IL-12.

In summary, IL23p19^(−/−) mice have attenuated in vivo T-cell responsesmanifesting in reduced DTH and humoral immune responses, andphenotypically resemble IL-17 deficient mice. Our results indicate thatclinical administration of IL-23 or its agonists might be beneficial tosupport T-cell function in immunization regimens and inimmunocompromised patients.

While the present invention has been described with reference to whatare considered to be the specific embodiments, it is to be understoodthat the invention is not limited to such embodiments. To the contrary,the invention is intended to cover various modifications and equivalentsincluded within the spirit and scope of the appended claims.

What is claimed is:
 1. A method for inhibiting interleukin-17 (IL-17)production by memory T cells, comprising administering to a mammaliansubject in need an interleukin-23 (IL-23) antagonist selected from thegroup consisting of an anti-IL-23 antibody, an anti-IL-23 receptorantibody, an immunoadhesin comprising an IL-23 subunit fused to animmunoglobulin constant region sequence or comprising an IL-23 receptorsubunit fused to an immunoglobulin constant region sequence, and ananti-sense oligonucleotide capable of inhibiting translation and/ortranscription of a gene encoding a subunit of native IL-23 polypeptideor capable of inhibiting translation and/or transcription of a geneencoding a subunit of a native IL-23 receptor polypeptide.
 2. The methodof claim 1, wherein the mammalian subject is a human.
 3. The method ofclaim 2, wherein said T cells are activated T cells.
 4. The method ofclaim 2, wherein said antagonist if an anti-IL-23 antibody or ananti-IL-23 receptor antibody.
 5. The method of claim 4, wherein saidantibody is an antigen-binding antibody fragment.
 6. The method of claim5, wherein said antibody fragment is selected from the group consistingof Fv, Fab, Fab′, and F(ab′)₂ fragments.
 7. The method of claim 4,wherein said antibody is a full-length antibody.
 8. The method of claim4, wherein said antibody is chimeric, humanized or human.